The overall goal of this study is to provide a more complete molecular understanding of how proline and proline metabolism impact redox-linked physiological processes. Proline metabolism is recognized as having an important impact on bioenergetics, cellular redox control, apoptosis, cancer, and schizophrenia. The mitochondrion enzyme proline dehydrogenase (PRODH) plays an important role in tumor suppression by generating proline-dependent reactive oxygen species (ROS) during p53-mediated apoptosis. A less well-recognized role of proline is its potential to protect against oxidative stress, a property that has been relatively unexplored in mammalian systems until only recently. Our central hypothesis is that proline functions as broad-based antioxidant in mammalian cells during stress by scavenging ROS and maintaining redox homeostasis. This hypothesis is supported by our strong preliminary results which show intracellular proline accumulation in mammalian cells correlates with decreased ROS levels and increased protection against oxidative stress. Our preliminary results also show that proline biosynthesis is upregulated under oxidative stress conditions, providing evidence that proline accumulation is a physiological stress response. While there is strong evidence that proline has an important role in cellular apoptotic pathways, the ability of proline to scavenge ROS and impact redox homeostasis has not been established. The main objectives of this project are to evaluate the protective function of proline against ROS mediated stress in various cell types and in vivo oxidative carcinogenesis. We also aim to elucidate the mechanisms whereby proline functions as a ROS scavenger and discover regulatory factors of proline metabolism under physiological stress conditions. The specific aims of this study are: 1. Determine the protective ability of endogenous proline against oxidative stress. 2. Elucidate the mechanism by which proline scavenges ROS. 3. Establish physiological significance of proline protection against oxidative stress. 4. Identify genes that regulate proline metabolism and ROS-induced cell death. Inborn errors in proline metabolic genes are manifested in neurological dysfunctions such as schizophrenia and increased incidence of seizures. A key enzyme in proline metabolism, proline dehydrogenase, is up- regulated by tumor suppressor p53 to generate reactive oxygen species that lead to cell death, thus helping to prevent cancer. This project will investigate how proline and proline metabolism modulate cellular reactive oxygen species and protect against oxidative stress. The results from this study will provide a molecular understanding for how proline metabolism contributes to cancer and mental illness and potentially suggest opportunities for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Inborn errors in proline metabolic genes are manifested in neurological dysfunctions such as schizophrenia and increased incidence of seizures. A key enzyme in proline metabolism, proline dehydrogenase, is upregulated by tumor suppressor p53 to generate reactive oxygen species that lead to cell death, thus helping to prevent cancer. This project will investigate how proline and proline metabolism modulate cellular reactive oxygen species and protect against oxidative stress. The results from this study will provide a molecular understanding for how proline metabolism contributes to cancer and mental illness and potentially suggest opportunities for therapeutic intervention.